The Role of MTHFR C677T Polymorphism on Blood Homocysteine Concentration and the Effect of Both of this on Susceptibility to Stroke

Overproduction of homocysteine exerts toxic effect on endothelial linage of blood vessels supplying blood into the brain and developing condition of Stroke. Methylenetetrahydrofolatereductase (MTHFR) plays a central role in regulation of homocysteine concentration in the cell. The (677C>T) polymorphic variant of MTHFR gene is known to hyperhomocysteinemia and hens 677C<T MTHFR variant may be a factor for stroke susceptibility. This study investigated whether the genetic variability in the MTHFR gene is related with overproduction of homocysteine as well as susceptibility for Stroke in the central Indian population. A total of 100 medically certified Stroke patients (case) and 223 healthy subjects (control) were recruited from central India as a sample for this investigation. We found a significant (P<0.0001) deference in mean values of tHyc between case and control. ‘T’ allele was present in higher proportion in Stroke patients (P=0.06) as compared with Healthy control group. We concluded that the homozygous MTHFR 677T condition made moderate susceptibility for hyperhomocysteinemia and Stroke.


Introduction
Stroke is leading cause of death among cardiovascular diseases and most prevalent factor for disability in world population [1]. Stroke arises in most of the chances by damaging internal endothelial lining of the blood vessels. The homocysteine exert its toxic effect on damaged endothelium by enhanced lipid peroxidation and generation of free radicals result into inflammation [2]. Due to developing inflammation, artery gets choked leading to partial to complete blockage of blood supply to the respective organ. An increased homocysteine in the blood is thus related with acute endothelial dysfunction [3]. The excess homocysteine is remethyleted into methionine and this step is catalyzed by Methionine synthase, which uses B12 as coenzyme and methylene-tetrahydrofolate (MTHF) as substrate. The formation of MTHF from tetrahydrofolate is catalyzed by Methylene-tetrahydrofolate reductase (MTHFR) [4]. The C677T mutation of the MTHFR gene, which leads to the synthesis of a thermolabile form of MTHFR that is responsible for 50% of the MTHFR activity [5]. is based on a novel assay principle that assesses the co-substrate conversion product (a molecule that is not a substrate of the Hcy conversion enzyme.

Materials and Methods
In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co-substrate, S-adenosylo-methionine (SAM) catalyzed by a Hcy S-methyltransferase to form methionine (the Hcy conversion product of Hcy) and S-adenosylhomocysteine (SAH, the co-substrate conversion product). SAH is assessed by coupled enzyme reactions including SAH hydrolase, adenosine (Ado) deaminase and glutamate dehydrogenase, wherein SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase. The formed Hcy that is originated from the co-substrate SAM is cycled into the Hcy conversion reaction by Hcy S-methyltransferase. This forms a co-substrate conversion product based enzyme cycling reaction system with significant amplification of detection signals. The formed Ado is immediately hydrolyzed into inosine and ammonia which reacts with glutamate dehydrogenase with concomitant con-versions of NADH to NAD+. The concentration of Hcy in the sample is indirectly proportional to the amount of NADH converted to NAD+ (ΔA340nm).

DNA isolation and Quantification
Genomic DNA was extracted from whole blood by the modification of salting out procedure described by Miller and coworkers [6]. The integrity of high molecular weight DNA is an important factor, which should be considered during extraction steps. Integrity was checked by electrophoresis on 0.8%. The high molecular weight genomic DNA appeared as a single band near the well. DNA was quantified by measuring the optical density at 260nm. 5 μl of stock genomic DNA was taken and 995 μl of water was added (Dilution factor D.F. = 200), mixed well and OD was taken at 260 nm in a spectrophotometer (Systronic, India).

The Detection of MTHFR C677T Polymorphism
The MTHFR C677T polymorphism was sought using a PCR-RFLP method.
The transition of C→T at the position 677 produces restriction site for HinfI, by which the polymorphic MTHFR gene and wild type gene can be identified after PCR amplification, restriction digestion and gel electrophoresis.
Primers: Primers were synthesized by Sigma Aldrich, India and amplification was carried out using MJ research thermolcycler those Statistical Analysis: We analyzed data by Fischer's exact test, unpaired t-test, and Odds ratio with 95% confidence interval and interpret results for P value.

Results
The demographic parameters of study groups are presented in (   Table 2. This study found significant differences (P<0.0002) in Homocysteine level between case and control group carrying same genotypes ( Figure 1).
These observations suggest the mutation in MTHFR is related with hyperhomocysteinemia. To observe role of mutation in MTHFR to susceptibility for stroke we analyzed the distribution of wild and mutant genotypes and allele between patients and control.
The distribution of genotypes and alleles are statistically examined by Fischer exact test for significant difference and the values are given in  Table 3 The distribution of genotypes and alleles between Stroke patients and control are statistically examined by Fischer exact test for significant difference The P values after Bonferroni Correction.

Discussion
We have provided statistical evidence that the MTHFR 677C >T  MTHFR C677T alleles shown a weak but significant interaction with disease and found low P value for Stroke but strong significantly associated with hyperhomocysteinemia. These findings are consistent with Indian studies [8,9] but in Japanese population only women associated with disease susceptibility [10].
Several studies on North Indian population were found significant association of MTHFR genotype with hyperhomocysteinemia but not with Stroke [11].
A study from west Bengal has found no significant correlation between the studied factors (hyperhomocysteinemia, TT and CT genotypes) and single vs recurrent stroke [12]. South Indian population has shown slightly increased frequency of CT genotype which is significantly associated with hyperhomocysteinemia as well as myocardial infarction [13]. In eastern Indian population MTHFR genotype is not significantly associated especially with Stroke but found in positive correlation with hyperhomocysteinemia [14,15].